Biochemical, Antioxidant Properties and Antimicrobial Activity of Epiphytic Leafy Liverwort Frullania dilatata (L.) Dumort

In this study, the biochemical, antioxidant properties, and antimicrobial activity of the epiphytic leafy liverwort Frullania dilatata (L.) Dumort were investigated. Due to the scarcity and difficulty in obtaining liverworts, research on their bioactivity is limited; thus, this study aimed to uncover the potential of F. dilatata. The antimicrobial activity was evaluated against various microorganisms, including food isolates, clinical isolates, multidrug-resistant strains, and standard strains, using the disk diffusion method and determining the minimum inhibitory concentration (MIC) values. This study represents the first antioxidant investigation on F. dilatata and an antimicrobial study using ethanol extract and the disk diffusion method. Notably, susceptibility was observed in Enterococcus faecalis ATCC 29212, Enterococcus faecium FI, Listeria monocytogenes ATCC 7644, Providencia rustigianii MDR, and Staphylococcus aureus ATCC 25923. The antioxidant capacity was assessed using the DPPH method, emphasizing the high scavenging performance. Gas chromatography-mass spectrometry (GC-MS) analysis identified the primary compounds as frullanolide (19.08%), 2,3-Dimethylanisole (15.21%), linoleic acid (11.11%), palmitic acid (9.83%), and valerenic acid (5.3%). The results demonstrated the significant antimicrobial activity of F. dilatata against the tested microorganisms and its potent antioxidant properties. These findings emphasize the potential of F. dilatata as a promising source of natural antimicrobial and antioxidant agents, underscoring the importance of further investigation into its bioactive compounds and elucidating the mechanisms of action in future studies.


Introduction
Liverworts (Marchantiophyta) are a group of non-vascular plants that comprise over 9000 species, representing an important component of the bryophyte division [1]. These plants have a simple structure and possess a wide range of secondary metabolites, including terpenoids, phenolic compounds, and flavonoids, which contribute to their diverse biological activities [2]. Although liverworts are not generally included in mammalian diets, including those of humans, mainly due to their unpalatability and potential allergenic effects, they have been traditionally used in ethnomedicine for treating various ailments such as wounds, digestive disorders, and respiratory issues [3]. Recent scientific investigations have also revealed the potential of liverworts as a source of biologically active compounds with antimicrobial, antioxidant, anti-inflammatory, and anticancer properties. As a result, liverworts have attracted increasing attention from researchers who seek to explore their potential applications in modern medicine and pharmacology [4].
The minimum inhibitory concentration (MIC) values for the ethanol and water extracts of F. dilatata were determined against 13 bacterial and 2 yeast strains, as presented in Table 2. MIC values for the ethanol extract ranged from 0.1285 to 1.028 mg/mL, while the water extract displayed MIC values of 21.44 mg/mL against two of the tested microorganisms. No inhibitory effect was observed for several microorganisms in both the ethanol and water extracts. As all the parallels yielded the same results, the ANOVA test was deemed unnecessary, and the p-value is considered to be 1.
The antioxidant activity of the F. dilatata ethanol extract was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The analysis revealed that the extract exhibited concentration-dependent antioxidant activity. Ascorbic acid was used as a positive control and for comparison purposes. For the negative controls, ethanol without the extract was used. The results of the antioxidant activity are presented in Table 3 below. The DPPH radical scavenging assay revealed that the EC 50 value for F. dilatata was 0.03169 mg/mL. An ANOVA test was performed to determine the significance of differences between the parallels, yielding a p-value of 0.997492. As the p-value is greater than 0.05, it can be concluded that there is no significant difference between the parallels, indicating that the experimental results are reliable and consistent. Furthermore, the Pearson correlation coefficient was calculated to be 0.8690298, indicating a very strong positive correlation between the concentration of the extract and the DPPH scavenging activity. An additional ANOVA test was conducted to compare the difference in DPPH activity between the extract and ascorbic acid, resulting in a p-value of 0.1754. This indicates that the difference between the effects of ascorbic acid and the extract on DPPH activity is statistically insignificant.  FD: F. dilatata scavenging AA: ascorbic acid scavenging. The t-value represents the test statistic from the independent two-sample t-test comparing FD and AA at each concentration. The p-value is the probability of observing a t-value as extreme or more extreme than the calculated one under the null hypothesis, assuming no significant difference between the means. A smaller p-value (typically below 0.05) indicates a statistically significant difference. The ANOVA p-value between the three parallels was 0.997492, and the Pearson correlation coefficient was 0.8690298. When evaluating FD and AA across all concentrations using ANOVA, the p-value was 0.1754.
The chemical content of F. dilatata's ethanol extract was analyzed via GC/MS. Chemicals constituting more than 0.5% were considered the main components and are listed in Table 4, where each compound's chemical structure, chemical formula, molecular weight, and known activities are provided in the respective columns.

Discussion
The antimicrobial activity study for F. dilatata was previously performed only by Nikolajeva et al. [19]. In the study conducted by Nikolajeva et al., the antimicrobial activity of the water extract of F. dilatata against the S. aureus strain was determined using the minimum inhibitory concentration method (MIC). In our study, we also performed the MIC assay to evaluate the antimicrobial activity of both the ethanol and water extracts of F. dilatata against S. aureus. We obtained consistent results with this previous research when examining the single strain of S. aureus. Our MIC results showed that the ethanol extract had an effective concentration of 0.257 mg/mL against S. aureus ATCC25923, while the water extract had an effective concentration of 21.44 mg/mL against the same strain. These findings revealed that the ethanol extract was much more potent and effective at significantly lower concentrations compared to the water extract, which is consistent with the results obtained for the water extract in the previous study [19]. In our investigation, the largest inhibition zone of 11 mm was detected against the S. aureus ATCC 25923 strain using the disk diffusion method for the ethanol extract. S. aureus is found in the human flora, primarily located in the nose and skin of most healthy individuals, and is an important human pathogen that causes various clinical manifestations. This bacterium is a significant cause of hospital-acquired infections, leading to complications such as surgical site infections, bloodstream infections, and pneumonia. Additionally, S. aureus is associated with a wide range of health issues, including skin and soft tissue infections, food poisoning, and more severe cases such as endocarditis and sepsis. The increasing prevalence of antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA), highlights the urgent need for effective prevention and control strategies to mitigate the impact of this pathogen on public health [31]. Given the significant impact of S. aureus on public health and the increasing prevalence of antibiotic-resistant strains, our findings demonstrating the antimicrobial activity of F. dilatata against the S. aureus ATCC 25923 strain highlight the potential of this liverwort as a valuable source for developing alternative therapeutic agents.
There is a significant increase in bacterial drug resistance worldwide. By 2050, infectious diseases caused by MDR bacteria are expected to cause more deaths than cancer [32]. P. rustigianii is an invasive enteric MDR pathogen that can colonize the human intestinal tract, be isolated from human feces, and cause diarrhea [33]. In our study, using the disk diffusion method for the ethanol extract, we observed a 9 mm activity against P. rustigianii, and according to the MIC results for the ethanol extract, the effective concentration was 1.028 mg/mL. The limited bioactivity research on this bacterium means we still have much to learn about its properties. On the other hand, finding an effect against an MDR strain is of considerable importance. As research progresses, it will be crucial to understand the mechanism of efficacy against not only P. rustigianii but also other MDR bacteria in the future.
Listeria monocytogenes is a foodborne bacterium with significant pathogenic activity. It causes serious diseases, including listeriosis, characterized by symptoms such as fever, muscle aches, and gastrointestinal distress. In more severe cases, listeriosis can lead to meningitis, septicemia, and even death, particularly among vulnerable populations such as pregnant women, newborns, the elderly, and immunocompromised individuals [34,35]. The discovery of new alternative compounds against listeriosis becomes critical for the food industry, which is of great importance globally, as the incidence of listeriosis continues to rise and antibiotic resistance becomes more prevalent. The disk diffusion assay conducted in this research demonstrated a 9 mm inhibition zone against L. monocytogenes, indicating the potential of F. dilatata to serve as a source of new antimicrobial agents. Additionally, the MIC results revealed an effective concentration of 0.257 mg/mL for the ethanol extract and 21.44 mg/mL for the water extract against L. monocytogenes. These results are particularly promising considering the urgent need for novel approaches to combat L. monocytogenes and the increasing challenge of antibiotic resistance. Further research is necessary to elucidate the specific compounds responsible for this activity and to investigate their mechanisms of action, as well as to evaluate their potential for development into effective therapeutic agents against listeriosis and other related diseases.
Our study revealed that F. dilatata demonstrated considerable antimicrobial effects against both E. faecalis ATCC 29212 and E. faecium FI, showing zone diameters of 9 mm and 8 mm, respectively, in the disk diffusion assay for the ethanol extract. The MIC results for the ethanol extract against these strains were 0.1285 mg/mL for E. faecalis ATCC 29212 and 1.028 mg/mL for E. faecium FI. The impact on E. faecium FI is of particular importance, given the role of E. faecium in infections associated with healthcare settings. According to the World Health Organization (WHO), such infections affect a significant proportion of hospitalized patients in high-income countries, leading to increased morbidity and mortality rates [36]. Both E. faecalis and E. faecium are recognized as leading causes of infections in healthcare environments, with vancomycin-resistant E. faecium (VREfm) posing a serious threat to immunocompromised patients [37]. The growing prevalence of vancomycin resistance among E. faecium clinical isolates highlights the urgent need for alternative therapeutic options. The observed antimicrobial activity of F. dilatata against both E. faecalis ATCC 29212 and E. faecium FI in our study could potentially contribute to the discovery of new treatment strategies for tackling infections caused by these pathogens in healthcare settings.
In our investigation, we assessed the antifungal activity against two yeast strains, C. albicans DSMZ 1386 and C. tropicalis CI, using the MIC method for the ethanol extract. We obtained MIC results of 0.257 mg/mL for C. albicans DSMZ 1386 and 0.514 mg/mL for C. tropicalis CI. While no inhibitory effects were observed in the disk diffusion assay, the presence of antifungal activity in the MIC results might be attributed to the hydrophobic nature of the antifungal active compounds present in the extract. This hydrophobicity could potentially hinder diffusion in the agar medium or cause the compounds to evaporate before diffusing from the disk due to their high volatility.
Although the antimicrobial activity observed in the disk diffusion assay was not found to be extremely high, the fact that the plant studied is a liverwort. It, therefore, has a limited amount of plant material available for extraction, making the observed effect quite significant, even if it appears to be low. In contrast, the MIC results for the ethanol extract showed potent antimicrobial activity at relatively low concentrations against various strains, including yeasts. Moreover, the MIC results for the ethanol extract were consistently more effective at lower concentrations compared to the water extract for many strains.
This study is the first to reveal the antioxidant activity of F. dilatata. As a result of the antioxidant activity study performed using the DPPH method, it was determined that the ethanol extract of F. dilatata exhibited a remarkable effect of 89.294% at a concentration of 200 µg/mL. This result is notably close to the 94.515% effect of ascorbic acid, a well-known antioxidant agent, at the same concentration. The strong antioxidant properties exhibited by F. dilatata, nearly rivaling those of ascorbic acid, highlight its potential as a valuable natural source of antioxidants.
F. dilatata extract, which has been analyzed using gas chromatography-mass spectrometry (GC-MS), has been found to contain beta-Elemene and linoleic acid as its major components. Previous studies have reported that both of these compounds possess notable antioxidant properties [20,30]. Antioxidants are essential in the body's defense against oxidative stress, which is responsible for the damage caused by free radicals. The presence of these compounds in F. dilatata suggests that the extract has potential therapeutic value in combating oxidative stress-related diseases.
The beta-Elemene, a sesquiterpene compound, has been previously associated with various biological activities, including anti-inflammatory, antitumor, and immunomodulatory effects [20]. Its antioxidant properties contribute to its overall therapeutic potential, and its presence in F. dilatata extract may be responsible for a portion of the observed antioxidant activity.
Similarly, Linoleic acid, an essential polyunsaturated fatty acid, has been known to play a crucial role in various physiological processes, including cellular signaling, inflammation regulation, and membrane structure maintenance [30]. Its antioxidant properties help protect cellular components from oxidative damage, which could, in turn, help maintain overall cellular health and function.
The findings from this study suggest that the antioxidant activity observed in F. dilatata extract can be attributed, at least in part, to the presence of beta-Elemene and linoleic acid. These compounds may work synergistically to provide potent antioxidant effects, making F. dilatata a potentially valuable natural resource for the development of new antioxidant agents or supplements. Further research is necessary to elucidate the precise mechanisms through which these compounds exert their antioxidant effects and to explore the full range of potential applications of F. dilatata extract in promoting health and well-being.
Frullania species are notable as liverworts that cause very intense allergenic contact dermatitis. F. dilatata used in this study is one of the species that causes allergenic contact dermatitis. As a result of GC-MS analysis of F. dilatata extract, it was determined that it contains Frullanolide in its structure. This substance is a sesquiterpene lactone and is known to cause allergenic contact dermatitis [38]. As a result of the study conducted by Chimplee et al. [24], it was determined that the same substance showed anti-breast cancer activity.
The antimicrobial and antioxidant activity results of F. dilatata are consistent with the compounds identified through GC/MS analysis and the known bioactivity properties of these compounds. However, it is also possible that the activity may be influenced by unidentified compounds not found in the universal libraries. Further studies should investigate the identification and activity of these unknown compounds, particularly in terms of their synergistic and antagonistic effects on bioactivity.

Plant Sample
The liverwort F. dilatata utilized in this study was collected from Canakkale Kazdaglari, specifically in the Yenice section of the Kazdagları Mountains at the coordinates 39 • 55 50.80 N, 27 • 12 42.60 E, and an altitude of 356 m. The collection took place on 22 June 2021 within a 30-m diameter area. The substrate in the area consisted of Pinus nigra subsp. pallasiana (Lamb.) Holmboe (Anatolian black pine) contributed to the formation of a microecosystem that allowed for the dense growth of the species due to the high moisture content in the region. Sustainable collection practices were employed, ensuring the continuity of the population without causing any significant damage to the natural habitat.
During the collection process, care was taken to obtain samples from the same substrate at the same time. After collection, the liverwort samples were dried in a shaded area, away from direct sunlight, and then stored in a dark and cool environment to preserve their properties until they were used in the experiments. The liverwort samples were identified by Dr. Özcan Simsek, and voucher specimens have been preserved at the Dokuz Eylul University's Research and Application Center for Fauna Flora (FAMER).
This study was conducted under the supervision and with the permission of the Turkish Ministry of Agriculture and Forestry, General Directorate of Nature Conservation and National Parks (permit number E-21264211-288.04-3471957).

Extraction Process
The extraction method for both ethanol and water extracts involved the following steps: After the drying process, the plant samples were ground to a fine powder using a grinder. For the ethanolic extract, 10 g of powdered sample were mixed with 200 mL of absolute ethyl alcohol (Sigma-Aldrich, Saint Louis, MO, USA), while for the water extract, 5 g of powdered sample were combined with 100 mL of ultra-pure water. Both samples were then shaken at 140 rpm at room temperature for 96 h, instead of the standard 48 or 72 h typically preferred for seed plants, to obtain a higher yield of compounds. After shaking, both the ethanolic and water extracts were filtered through Whatman No. 1 filter paper, primarily for the removal of solid residues. The filtrates were collected in evaporation flasks.
For the ethanolic extract, the extraction solvent was evaporated under a vacuum using a rotary evaporator (Buchi R3, BÜCHI, Labortechnik AG, Postfach, Flawil, Switzerland) at 30 • C. The dried crude ethanolic extract was collected using an analytical balance for accurate weighing and used to prepare the extracted stock, yielding 0.09 g in 35 mL (2.57 mg/mL). The ethanolic extract stock was stored at +4 • C in a refrigerator until the time of application. For the MIC test, the dried crude ethanolic extract was dissolved in DMSO at a non-toxic concentration of 1%. In addition, a 1% DMSO solution was used as a negative control to ensure no toxic effect on the microorganisms during the experiment.
The water extract was dried using a HyperCOOL HC3110 lyophilizer (Gyrozen, Gimpo, Korea). The water extract yielded 0.536 g in 10 mL (53.6 mg/mL), and the final stock was prepared and passed through a 0.45 µm Minipore membrane filter to ensure sterility. The water extract stock was stored at −35 • C in a deep freezer until the time of application [15].

Microorganisms
In this study, we used 8 standard, 1 clinical isolate, 3 multidrug resistant, and 1 food-isolated bacteria, as well as 1 standard and 1 clinical isolated yeast strain. The standard gram-positive bacteria included E. faecalis ATCC 29212, L. monocytogenes ATCC 7644, S. aureus ATCC 25923, and S. hominis ATCC 27844. The standard gram-negative bacteria comprised E. coli ATCC 25922, P. aeruginosa DSMZ 50071, S. typhimurium SL1344, and A. baumannii CECT 9111. The gram-negative clinically isolated bacterium was K. pneumoniae. The gram-positive multidrug-resistant strain was S. pneumoniae, while the gram-negative multidrug-resistant strains included P. rustigianii and A. baumannii. The gram-positive food-isolated bacterium was E. faecium. The standard yeast was C. albicans DSMZ 1386, and the clinically isolated yeast was C. tropicalis. The strains were obtained from the Department of Biology at Dokuz Eylül University's Faculty of Science (İzmir, Türkiye).

Microorganism Inoculum Preparation
Bacterial strains used to determine the antimicrobial activity of F. dilatata ethanol extract were incubated at 37 • C for 24 h, while yeast strains were incubated at 27 • C for 48 h. The inocula of bacterial strains were adjusted to 0.5 McFarland in a sterile 0.9% NaCl solution, containing approximately 10 8 CFU·mL −1 for bacterial strains and 10 7 CFU·mL −1 for yeast strains [15].

Antimicrobial Assay
The antimicrobial activity of F. dilatata ethanol extract was assessed using both the disk diffusion test and the MIC determination, while the water extract was evaluated solely through the MIC method due to testing limitations. The disk diffusion test for the ethanol extract followed the protocol described by Andrews [39]. Initially, Mueller Hinton Agar (BD Difco, USA) was poured into a 90 mm sterile Petri dish to a thickness of 4.0 mm ± 0.5 mm. The ethanol extract of F. dilatata was applied to 6 mm Oxoid antimicrobial susceptibility test disks in quantities of 50, 100, and 200 µL. The ethyl alcohol on the disks, which might affect the test results, was allowed to evaporate by drying overnight at 30 • C under sterile conditions. Subsequently, the culture media were inoculated with the previously prepared inoculum. In the final step, the extract-loaded disks were placed on the media and incubated. At the end of the incubation period, the diameters of the inhibition zones formed around the disks were measured and recorded in millimeters [15]. Gentamicin was used as a positive control, and its results are also included in the Table 1. Disks impregnated with 200 µL of absolute ethanol, without the extract, served as the negative controls. The ethanol was allowed to evaporate under the same conditions and duration as in the experimental procedure, resulting in no observed inhibition zones.
The minimum inhibitory concentration (MIC) values for the F. dilatata ethanol and water extracts were ascertained utilizing the broth microdilution method, following the approach presented by Kowalska-Krochmal and Dudek-Wicher [40]. Mueller-Hinton broth (MHB) was the chosen medium for the cultivation of various microbial strains. The cell density for each reference strain solution was adjusted to correspond to the 0.5 McFarland standard (1.5 × 10 8 CFU/mL). A range of dilutions for the plant materials was prepared, and 100 µL from each dilution was pipetted into the wells of a 96-well plate. Subsequently, 50 µL of the microbial inoculum was introduced to achieve a final volume of 100 µL per well. Microbial growth was assessed by spectrophotometrically supported visual examination. The positive control comprised MHB containing the test microorganisms. The MIC corresponds to the lowest concentration of the plant sample required to inhibit bacterial growth after incubation for 24 h. The findings were presented in mg/mL, and the experiments were conducted in triplicate for accuracy.

Antioxidant Activity by DPPH Radical Scavenging Method
The antioxidant activity of F. dilatata ethanol extract was determined using the DPPH method, which is based on measuring the extract's antioxidant capacity to scavenge DPPH radicals. The DPPH solution for the study was prepared by adding 3.9432 mg of DPPH (2,2diphenyl-1-picrylhydrazyl) to 50 mL of ethanol and shielding it from light [41]. The DPPH solvent was added to the F. dilatata extract and incubated for 30 min at room temperature in a dark environment. After completing the incubation, the absorbance at 515 nm was measured using a spectrophotometer (Biotek Microplate Spectrophotometer, Winooski, VT, USA). Ascorbic acid served as a positive control for the DPPH test [42]. For the negative controls, ethanol without the extract was used.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Gas chromatography/mass spectrometry (GC/MS) is a combination of two analytical techniques: gas chromatography separates the components in a mixture, while mass spectrometry defines the components structurally [43]. In this study, an Agilent model 8890 GC/MS instrument was used. The injector temperature was set at 350 • C, and helium gas served as the carrier gas (1 mL/min). The injection mode was a 10:1 split, with an injection volume of 1 µL. The oven temperature was programmed to increase from 40 • C to 150 • C at a rate of 4 degrees per minute, from 150 • C to 180 • C at 3 degrees per minute, from 180 • C to 230 • C at 2 degrees per minute, and from 230 • C to 280 • C at 1 degree per minute [15]. The ions formed as a result of electron ionization using the GC/MS technique were separated according to their mass-to-charge ratio and recorded in the detector. The data were then collected from the computer. The compounds were identified by matching them with the data of compounds in the latest NIST and Wiley data libraries.

Statistical Analysis
All experiments were conducted in triplicate. A one-way ANOVA was employed for the statistical analysis, with a significance level set at 0.05.

Conclusions
This study highlights the promising antimicrobial and antioxidant potential of F. dilatata ethanol extract. Our results demonstrated significant antimicrobial activity against various bacterial strains, including Gram-positive and Gram-negative bacteria, as well as multidrug resistant strains. In addition, the extract exhibited moderate antioxidant activity. The GC-MS analysis revealed the presence of several bioactive compounds, which may be responsible for the observed activities. However, the possibility of undiscovered compounds and their synergistic or antagonistic effects cannot be ruled out. Future studies should focus on the identification of these compounds and their specific contributions to the extract's bioactivity. Furthermore, investigations into the extract's mode of action and its potential for development into novel therapeutic agents are warranted. The findings of this study contribute to the growing body of evidence supporting the potential of liverwortderived compounds, which may pave the way for the development of synthetic production methods for these bioactive molecules in the pharmaceutical and food industries.
In addition to the promising antimicrobial and antioxidant potential of F. dilatata ethanol extract, the GC-MS analysis identified Frullanolide as a primary bioactive compound, which is known for its anticancer properties. This suggests that future studies may investigate the potential anticancer effects of F. dilatata extract, specifically focusing on Frullanolide.  Data Availability Statement: All data that were generated or analyzed during this study have been included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.